Selective mcl-1 binding peptides

ABSTRACT

Provided herein are peptides that bind Mcl-1. Also provided are compositions containing these polypeptides and methods of using such peptides in the treatment of cancer that include administering to a subject one of the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/248,987, filed on Oct. 30, 2015. The entire contents of theforegoing are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to peptides that bind Mcl-1 and methods of usingsuch peptides in the treatment of cancer.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an ASCII text file named Sequence Listing. The ASCIItext file, created on Aug. 2, 2021, is 18 kilobytes in size. Thematerial in the ASCII text file is hereby incorporated by reference inits entirety.

BACKGROUND

Mcl-1 is one of the most frequently amplified genes in cancers and animportant factor in resistance to chemotherapeutic agents. Mcl-1 is amember of a family of anti-apoptotic proteins that have homology toBcl-2 and contain a so-called BH3 domain. Mcl-1 and others members ofthe family (e.g., Bcl-xL, Bcl-2, Bcl-w, Bfl-1 and Bcl-b) block apoptosisby interfering with the homo-oligomerizing of Bak and Bax. Theanti-apoptotic proteins either bind directly to Bax and Bak or bindrelated pro-apoptotic activator proteins (Bim, Bid and Puma), preventingactivation of Bax and Bak. Other proteins having BH3-domain, calledsensitizers, antagonize anti-apoptotic function by binding competitivelywith Bax/Bak and activators.

Agents that selectively bind Mcl-1 compared to other members of theBcl-2 family of anti-apoptotic proteins, such as Bcl-xL or Bcl-2, may beuseful in treating a variety of cancers.

SUMMARY

The present disclosure describes peptides mimicking BH3 motifs that bindhuman Mcl-1. The peptides are relatively selective for binding Mcl-1 inthat they bind human Mcl-1 with greater affinity than they bind one ormore of several proteins considered human homologs of Bcl-1, forexample, Bfl-1, Bcl-w, Bcl-xL and Bcl-2.

In some aspects, the present disclosure provides a compound comprising,consisting of, or consisting essentially of the amino acid sequence 1F1G 2A 2B 2C 2D 2E 2F 2G 3A 3B 3C 3D 3E 3F 3G 4A 4B 4C 4D 4E 4F 4G 5A(SEQ ID NO: 1), wherein 1F is R or a conservative substitution or ismissing; 1G is P or a conservative substitution or is missing; 2A is Eor a conservative substitution or is missing; 2B is I or a conservativesubstitution; 2C is W or a conservative substitution; 2D is M or aconservative substitution, or norleucine (B); 2E is T or a conservativesubstitution, V or a conservative substitution, 2-aminoisobutyric acid(Aib), or X; 2F is Q or a conservative substitution; 2G is G or aconservative substitution; 3A is L or a conservative substitution, F ora conservative substitution, pentafluoro phenylalanine, cyclohexylalanine (Cha), or homo-cyclohexyl alanine (H-Cha); 3B is R or aconservative substitution, W or a conservative substitution, Q or aconservative substitution, D or a conservative substitution, Y or aconservative substitution, Aib, D-phenyl glycine, α,α methyl leucine,α,α methyl phenylalanine, or X; 3C is R or a conservative substitution;3D is L or a conservative substitution; 3E is G or a conservativesubstitution; 3F is D or a conservative substitution; 3G is E or aconservative substitution; 4A is I or a conservative substitution; 4B isN or a conservative substitution, or X; 4C is A or a conservativesubstitution; 4D is Y or a conservative substitution; 4E is Y or aconservative substitution; 4F is A or a conservative substitution, or X;4G is R or a conservative substitution or is missing; 5A is R or aconservative or is missing; provided that 2E, 3A, 3B, 4B and 4F are notT, L, R, N and A respectively, wherein X is an amino acid whose sidechain is replaced with an intermolecular link to another amino acid. Insome cases 1F, 1G and 2A are missing. In some cases, 2D, 4B and 4F areB, N and A respectively.

In some aspects, the present disclosure provides a compound comprisingthe amino acid sequence 1F 1G 2A 2B 2C 2D 2E 2F 2G 3A 3B 3C 3D 3E 3F 3G4A 4B 4C 4D 4E 4F 4G 5A (SEQ ID NO: 1), wherein 1F is R or aconservative substitution or is missing; 1G is P or a conservativesubstitution or is missing; 2A is E or a conservative substitution or ismissing; 2B is I or a conservative substitution; 2C is W or aconservative substitution; 2D is M or a conservative substitution, ornorleucine (B); 2E is T or a conservative substitution, V or aconservative substitution, 2-aminoisobutyric acid (Aib), or X; 2F is Qor a conservative substitution; 2G is G or a conservative substitution;3A is L or a conservative substitution, F or a conservativesubstitution, pentafluoro phenylalanine, cyclohexyl alanine (Cha), orhomo-cyclohexyl alanine (H-Cha); 3B is R or a conservative substitution,W or a conservative substitution, Q or a conservative substitution, D ora conservative substitution, Y or a conservative substitution, Aib,D-phenyl glycine, α,α methyl leucine, α,α methyl phenylalanine, or X; 3Cis R or a conservative substitution; 3D is L or a conservativesubstitution; 3E is G or a conservative substitution; 3F is D or aconservative substitution; 3G is E or a conservative substitution; 4A isI or a conservative substitution; 4B is N or a conservativesubstitution, or X; 4C is A or a conservative substitution; 4D is Y or aconservative substitution; 4E is Y or a conservative substitution; 4F isA or a conservative substitution, or X; 4G is R or a conservativesubstitution or is missing; 5A is R or a conservative or is missing;provided that 2E, 3A, 3B, 4B and 4F are not T, L, R, N and Arespectively, and wherein the side chains of two amino acids separatedby 3 or 6 amino acids are optionally replaced by an intramolecularcross-link. In some cases 1F, 1G and 2A are missing. In some cases, 2D,4B and 4F are B, N and A respectively.

In some cases the compound comprises at least one amino acid that is notone of the 20 common, naturally-occurring amino acids at a positionselected from the group consisting of 2E, 3A, and 3B. For example, anamino acid that is not one of the 20 common, naturally-occurring aminoacids may be 4-hydroxyproline, α-4-pentenyl alanine, aminoisobutyricacid (Aib), cyclohexyl alanine (Cha), norleucine, desmosine,gamma-aminobutyric acid, beta-cyanoalanine, norvaline,4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine,1-amino-cyclopropanecarboxylic acid,1-amino-2-phenyl-cyclopropanecarboxylic acid,1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid,3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid,4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid,2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioicacid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta-and/para-substituted phenylalanines (e.g., substituted with —C(═O)C6H5;—CF3; —CN; -halo; —NO2; CH3), disubstituted phenylalanines, substitutedtyrosines (e.g., further substituted with -Q=0)C6H5; —CF3; —CN; -halo;—NO2; CH3), or statine. Additionally, the amino acids can be derivatizedto include amino acid residues that are hydroxylated, phosphorylated,sulfonated, acylated, and glycosylated.

In some embodiments, the compound comprises, consists of, or consistsessentially of the amino acid sequence IWBTQGChaRRLGDEINAYYARR (SEQ IDNO: 8), wherein up to 6 (e.g., 1, 2, 3, 4, 5 or 6) of the amino acidsare replaced by another amino acid. In some cases, no more than 2 of theamino acids are replaced by another amino acid. In some cases, none ofthe amino acids are replaced by another amino acid. In some cases thesubstitution is a conservative substitution. In some cases, neither Bnor Cha is substituted.

In some embodiments, the compound comprises, consists of, or consistsessentially of the amino acid sequence IWBTQGH-ChaRRLGDEINAYYARR (SEQ IDNO: 9), wherein up to 6 (e.g., 1, 2, 3, 4, 5 or 6) of the amino acidsare replaced by another amino acid. In some cases, no more than 2 of theamino acids are replaced by another amino acid. In some cases, none ofthe amino acids are replaced by another amino acid. In some cases, thesubstitution is a conservative substitution. In some cases, neither Bnor H-Cha is substituted

In some embodiments, the compound comprises, consists of, or consistsessentially of the amino acid sequence IWBAibQGLRRLGDEINAYYARR (SEQ IDNO: 10), wherein up to 6 (e.g., 1, 2, 3, 4, 5 or 6) of the amino acidsare replaced by another amino acid. In some cases, no more than 2 of theamino acids are replaced by another amino acid. In some cases, none ofthe amino acids are replaced by another amino acid. In some cases, thesubstitution is a conservative substitution. In some cases, neither Bnor Aib is substituted.

In some embodiments, the compound comprises, consists of, or consistsessentially of the amino acid sequence IWBAibQGChaRRLGDEINAYYARR (SEQ IDNO: 11), wherein up to 6 (e.g., 1, 2, 3, 4, 5 or 6) of the amino acidsare replaced by another amino acid. In some cases, no more than 2 of theamino acids are replaced by another amino acid. In some cases, none ofthe amino acids are replaced by another amino acid. In some cases, thesubstitution is a conservative substitution. In some cases, neither Bnor Cha is substituted.

In some embodiments, the compound comprises, consists of, or consistsessentially of the amino acid sequence IWBAibQGH-ChaRRLGDEINAYYARR (SEQID NO: 12), wherein up to 6 (e.g., 1, 2, 3, 4, 5 or 6) of the aminoacids are replaced by another amino acid. In some cases, no more than 2of the amino acids are replaced by another amino acid. In some cases,none of the amino acids are replaced by another amino acid. In somecases, the substitution is a conservative substitution. In some cases,H-Cha is not substituted

In some cases, the side chains of two amino acids separated by 3 or 6amino acids are replaced by an intermolecular crosslink. In some cases,the two amino acids are separated by 3 amino acids. In some cases, thetwo amino acids are separated by 6 amino acids.

In some cases the intermolecular crosslink is an alkylene or alkenylenegroup. In some cases, the intermolecular crosslink is an alkylene group.In some cases, the alkylene group is C7, C8, C9, C10, C11, C12 or C13.In some cases, the intermolecular crosslink is an alkenylene group. Insome cases, the alkenylene group is C7, C8, C9, C10, C11, C12 or C13.

In some cases, the intermolecular crosslink is a lactam bridge.

In some cases, the side chains of 4B and 4F form an intermolecularcrosslink. In some cases, the amino acid at 4B and the amino acid at 4Fare α-4-Pentenyl alanine.

In some cases, the compound comprises, consists of, or consistsessentially of the sequence IWBTQGLRRLGDEIXAYYXRR (SEQ ID NO: 2),wherein X is an amino acid whose side chain is replaced with anintermolecular link to another amino acid and wherein up to 6 (e.g., 1,2, 3, 4, 5 or 6) of the amino acids are replaced by another amino acid.In some cases, no more than two of the amino acids are replaced byanother amino acid. In some cases, none of the amino acids are replacedby another amino acid. In some cases, the substitution is a conservativesubstitution. In some cases, B is not substituted

In some cases, the compound comprises, consists of, or consistsessentially of the sequence IWBAibQGLRRLGDEIXAYYXRR (SEQ ID NO: 3),wherein X is an amino acid whose side chain is replaced with anintermolecular link to another amino acid and wherein up to 6 (e.g., 1,2, 3, 4, 5 or 6) of the amino acids are replaced by another amino acid.In some cases, no more than two of the amino acids are replaced byanother amino acid. In some cases, the substitution is a conservativesubstitution. In some cases, none of the amino acids are replaced byanother amino acid. In some cases, neither B nor Aib is substituted.

In some cases, the compound comprises, consists of, or consistsessentially of the sequence IWBAibQGChaRRLGDEIXAYYXRR (SEQ ID NO: 4),wherein X is an amino acid whose side chain is replaced with anintermolecular link to another amino acid and wherein up to 6 (e.g., 1,2, 3, 4, 5 or 6) of the amino acids are replaced by another amino acid.In some cases, no more than two of the amino acids are replaced byanother amino acid. In some cases, none of the amino acids are replacedby another amino acid. In some cases, none B, Aib and Char aresubstituted.

In some cases, the compound comprises, consists of, or consistsessentially of the sequence IWBAibQGLQRLGDEIXAYYXRR (SEQ ID NO: 5),wherein X is an amino acid whose side chain is replaced with anintermolecular link to another amino acid and wherein up to 6 (e.g., 1,2, 3, 4, 5 or 6) of the amino acids are replaced by another amino acid.In some cases, no more than two of the amino acids are replaced byanother amino acid. In some cases, none of the amino acids are replacedby another amino acid. In some cases, the substitution is a conservativesubstitution. In some cases, neither B nor Aib is substituted.

In some cases, the compound comprises, consists of, or consistsessentially of the sequence IWBAibQGLDRLGDEIXAYYXRR (SEQ ID NO: 6),wherein X is an amino acid whose side chain is replaced with anintermolecular link to another amino acid and wherein up to 6 (e.g., 1,2, 3, 4, 5 or 6) of the amino acids are replaced by another amino acid.In some cases, no more than two of the amino acids are replaced byanother amino acid. In some cases, none of the amino acids are replacedby another amino acid. In some cases, the substitution is a conservativesubstitution. In some cases, neither B nor Aib is substituted.

In some cases, the compound comprises, consists of, or consistsessentially of the sequence IWBTQGLQRLGDEIXAYYXRR (SEQ ID NO: 7),wherein X is an amino acid whose side chain is replaced with anintermolecular link to another amino acid and wherein up to 6 (e.g., 1,2, 3, 4, 5 or 6) of the amino acids are replaced by another amino acid.In some cases, no more than two of the amino acids are replaced byanother amino acid. In some cases, none of the amino acids are replacedby another amino acid. In some cases, the substitution is a conservativesubstitution. In some cases, B is not substituted.

In some cases of the compounds described herein, X is α-4-Pentenylalanine.

In some cases of the compounds described herein, at least one amino acidthat is not one of the 20 common, naturally-occurring amino acids isselected from the group consisting of: 4-hydroxyproline, α-4-pentenylalanine, aminoisobutyric acid (Aib), cyclohexyl alanine (Cha),norleucine, desmosine, gamma-aminobutyric acid, beta-cyanoalanine,norvaline, 4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine,N-methyl-L-leucine, 1-amino-cyclopropanecarboxylic acid,1-amino-2-phenyl-cyclopropanecarboxylic acid,1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid,3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid,4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid,2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioicacid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-,meta-substituted phenylalanines, para-substituted phenylalanines,disubstituted phenylalanines, substituted tyrosines and statine.

In some cases of the compounds described herein, the peptide includes ofno more than 24 amino acids. In some cases the peptide includes up to 50amino acids (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, etc.). In somecases, the compounds include at least one amino acid that is not one ofthe 20 common, naturally-occurring amino acids.

In some cases, the compounds described herein also comprise a detectablelabel. In some cases, the detectable label is linked to the peptide.

In some cases, the compounds described herein also comprise a moietylinked to the peptide. In some cases, the moiety can be a functionalpeptide, polyethylene glycol (PEG), alkyl groups (e.g., C1-C20 straightor branched alkyl groups), fatty acid radicals, and combinationsthereof. In some cases, the peptide is linked to PEG.

In some cases of the compounds described herein, the peptide is linkedto a second peptide or functional moiety. In some cases the secondpeptide or functional moiety modulates the activity of the compound. Forexample, in some cases the second peptide or functional moiety modulatesthe solubility of the compound, modulates the stability of the compound,modulates the ability of the compound to permeabilize a cell, acts totarget the compound within/to the cell, labels the compound, modulatesthe affinity of the compound for MCL-1 and/or other members of thefamily (e.g., Bcl-xL, Bcl-2, Bcl-w, Bfl-1 and Bcl-b), modulates thespecificity of the compound for MCL-1 and/or other members of the family(e.g., Bcl-xL, Bcl-2, Bcl-w, Bfl-1 and Bcl-b), and/or any combinationthereof. Modulating the activity of the compounds described herein canbe increasing or decreasing the activity.

In some cases of the compounds described herein, the peptides aremodified. In some cases, the modification is selected from the groupconsisting of acetylation, amidation, biotinylation, cinnamoylation,farnesylation, fluoresceination, formylation, myristoylation,palmitoylation, phosphorylation, stearoylation, succinylation,sulfurylation, and combinations thereof.

In some cases of the compounds described herein, the peptides include atleast one peptide bond that is replaced by a non-natural peptide bond.In some cases, the peptide bond is replaced by a bond selected from thegroup consisting of a retro-inverso bonds (C(O)—NH); a reduced amidebond (NH—CH2); a thiomethylene bond (S—CH2 or CH2-S); an oxomethylenebond (O—CH2 or CH2-O); an ethylene bond (CH2-CH2); a thioamide bond(C(S)—NH); a trans-olefin bond (CH═CH); a fluoro substitutedtrans-olefin bond (CF═CH); a ketomethylene bond (C(O)—CHR) or CHR—C(O)wherein R is H or CH3; and a fluoro-ketomethylene bond (C(O)—CFR orCFR—C(O) wherein R is H or F or CH3.

In some cases, the compounds described herein also comprise a carrierprotein.

In some aspects, the present disclosure provides a pharmaceuticalcomposition comprising the compounds described herein. In some aspects,the present disclosure provides a method of treating cancer comprisingadministering the compound described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 a-g|Results of circular dichroism, competition assays, BH3profiling, and stability assays.

FIG. 2 a-b|Results of competition fluorescence anisotropy bindingexperiments.

FIG. 3 a-c|Results of BH3 profiling assay of engineered MS1 and nativeBH3 peptides.

FIG. 4 a-b|Results of BH3 profiling assay of stapled BH3 peptides.

FIG. 5|Schematic of one bead one compound library formation.

FIG. 6|Sequences of the Mcl-1 binding peptides and structures ofuncommon amino acids.

FIG. 7|Results of circular dichroism to determine helicity.

FIG. 8|Results of competition assays to determine binding affinity andspecificity.

FIG. 9|Results of BH3 profiling assay of peptides with cell linesdependent on Mcl-1, Bcl-xL, Bcl-2, or Bfl-1 (1:Aib, 2-aminoisobutyricacid; 2: Cha, cyclohexylalanine; and 3: hCha, homo-cyclohexylalanine).

DETAILED DESCRIPTION

The present disclosure provides Mcl-1-binding peptides. In some cases,the peptides include amino acids other than the 20 common,naturally-occurring amino acids. In some cases the peptides have aninternal (intramolecular) cross-link (or staple) that replaces the sidechains of two amino acids that are separated by 3 or 6 amino acids. Insome cases the peptides have more than one internal cross-link (e.g.,the peptides include two staples or a stitch).

Amino acids are the building blocks of the peptides herein. The term“amino acid” refers to a molecule containing both an amino group and acarboxyl group as well as a side chain. Amino acids suitable forinclusion in the peptides disclosed herein include, without limitation,natural alpha-amino acids such as D- and L-isomers of the 20 commonnaturally-occurring alpha-amino acids found in peptides (e.g., Ala (A),Arg (R), Asn (N), Cys (C), Asp (D), Gln (Q), Glu (E), Gly (G), His (H),Ile (I), leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T),Trp (W), Tyr (Y), and Val (V), uncommon alpha-amino acids (including,but not limited to α,α-disubstituted and N-alkylated amino acids),common naturally-occurring beta-amino acids (e.g., beta-alanine), anduncommon beta-amino acids. Amino acids used in the construction ofpeptides of the present invention can be prepared by organic synthesis,or obtained by other routes, such as, for example, degradation of orisolation from a natural source.

There are many known amino acids beyond the 20 commonnaturally-occurring amino acids, any of which may be included in thepeptides of the present invention. Some examples of uncommon amino acidsare 4-hydroxyproline, α-4-pentenyl alanine, aminoisobutyric acid (Aib),cyclohexyl alanine (Cha), norleucine, desmosine, gamma-aminobutyricacid, beta-cyanoalanine, norvaline,4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine,1-amino-cyclopropanecarboxylic acid,1-amino-2-phenyl-cyclopropanecarboxylic acid,1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid,3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid,4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid,2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioicacid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta-and/para-substituted phenylalanines (e.g., substituted with —C(═O)C₆H₅;—CF₃; —CN; -halo; —NO2; CH3), disubstituted phenylalanines, substitutedtyrosines (e.g., further substituted with -Q=O)C₆H₅; —CF₃; —CN; -halo;—NO₂; CH₃), and statine. Additionally, amino acids can be derivatized toinclude amino acid residues that are hydroxylated, phosphorylated,sulfonated, acylated, and glycosylated, to name a few.

Useful amino acids include:

In some instances, peptides include only common, naturally-occurringamino acids, although uncommon amino acids (i.e., compounds that do notcommonly occur in nature but that can be incorporated into a polypeptidechain) and/or amino acid analogs as are known in the art mayalternatively be employed. Also, one or more of the amino acids in apeptide or polypeptide may be modified, for example, by the addition ofa chemical entity such as a carbohydrate group, a hydroxyl group, aphosphate group, a farnesyl group, an isofarnesyl group, a fatty acidgroup, a linker for conjugation, functionalization, or othermodification, etc.

In some instances, peptides can include (e.g., comprise, consistessentially of, or consist of) at least seven (e.g., 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) contiguous amino acids ofany of SEQ ID NOs: 1-12. In some instances, the peptides can comprisethe amino acid sequence of any of SEQ ID NOs: 1-12 with 1, 2, 3, 4, 5 or6 single amino acid substitutions, e.g., conservative substitutions. Insome instances, the peptides can comprise the amino acid sequence of anyof SEQ ID NOs: 1-12 and have the side chains of 2 amino acids separatedby 3 or 6 amino acids substituted by an internal cross-link and, inaddition to the cross-link, include 1, 2, 3, 4, 5 or 6 single amino acidsubstitutions, e.g., conservative substitutions. In some instances, thepeptides can comprise the amino acid sequence of any of SEQ ID NOs: 1-12and have the side chains of more than 2 amino acids separated by 3 or 6amino acids substituted by an internal cross-link and, in addition tothe cross-link, include 1, 2, 3, 4, 5 or 6 single amino acidsubstitutions, e.g., conservative substitutions.

In some instances, a “conservative amino acid substitution” can includesubstitutions in which one amino acid residue is replaced with anothernaturally-occurring amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

Methods for determining percent identity between amino acid sequencesare known in the art. For example, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, the length of a reference sequencealigned for comparison purposes is at least 70%, 80%, 90%, or 100% ofthe length of the reference sequence. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The determination of percent identitybetween two amino acid sequences is accomplished using the BLAST 2.0program. Sequence comparison is performed using an ungapped alignmentand using the default parameters (Blossom 62 matrix, gap existence costof 11, per residue gapped cost of 1, and a lambda ratio of 0.85). Themathematical algorithm used in BLAST programs is described in Altschulet al. (Nucleic Acids Res. 25:3389-3402, 1997).

As disclosed above, peptides herein include at least two modified aminoacids that together can form an internal (intramolecular) cross-link (orstaple), wherein the at least two modified amino acids are separated by:(A) three amino acid (i.e., i, i+4) or (B) six amino acids (i.e., i,i+7). In the case of a cross-between i and i+4 the cross-link can be aC8 alkylene or alkenylene. In the case of a cross-link between i and i+7the cross-link can be a C11, C12 or C13 alkylene or alkenylene. When thecross-link is an alkenylene there can one or more double bonds. In thecase of a cross-link between i and i+4 the cross-link can be a C8 alkylor alkene. In the case of a cross-link between i and i+7 the cross-linkcan be a C11, C12 or C13 alkyl or alkene (e.g., a C11 alkene having asingle double bond). When the cross-link is an alkene there can be oneor more double bonds.

“Peptide stapling” is a term coined from a synthetic methodology whereintwo olefin-containing side-chains (e.g., cross-linkable side chains)present in a polypeptide chain are covalently joined (e.g., “stapledtogether”) using a ring-closing metathesis (RCM) reaction to form across-linked ring (Blackwell et al., J. Org. Chem., 66: 5291-5302, 2001;Angew et al., Chem. Int. Ed. 37:3281, 1994). As used herein, the term“peptide stapling,” includes the joining of two (e.g., at least one pairof) double bond-containing side-chains, triple bond-containingside-chains, or double bond-containing and triple bond-containing sidechain, which may be present in a polypeptide chain, using any number ofreaction conditions and/or catalysts to facilitate such a reaction, toprovide a singly “stapled” polypeptide. The term “multiply stapled”polypeptides refers to those polypeptides containing more than oneindividual staple, and may contain two, three, or more independentstaples of various spacings and compositions. Additionally, the term“peptide stitching,” as used herein, refers to multiple and tandem“stapling” events in a single polypeptide chain to provide a “stitched”(e.g., tandem or multiply stapled) polypeptide, in which two staples,for example, are linked to a common residue. Peptide stitching isdisclosed in WO 2008121767 and in WO 2010/068684, which are both herebyincorporated by reference. In some instances, staples, as used herein,can retain the unsaturated bond or can be reduced (e.g., as mentionedbelow in the stitching paragraph description).

While many peptide staples have all hydrocarbon cross-links, other typeof cross-links or staples can be used. For example, triazole-containing(e.g., 1, 4 triazole or 1, 5 triazole) crosslinks can be used (Kawamotoet al. 2012 Journal of Medicinal Chemistry 55:1137; WO 2010/060112).

Stapling of a peptide using all-hydrocarbon cross-link has been shown tohelp maintain its native conformation and/or secondary structure,particularly under physiologically relevant conditions (Schafmiester etal., J. Am. Chem. Soc., 122:5891-5892, 2000; Walensky et al., Science,305:1466-1470, 2004).

Stapling the polypeptide herein by an all-hydrocarbon crosslinkpredisposed to have an alpha-helical secondary structure can constrainthe polypeptide to its native alpha-helical conformation. Theconstrained secondary structure may, for example, increase the peptide'sresistance to proteolytic cleavage, may increase the peptide's thermalstability, may increase the peptide's hydrophobicity, may allow forbetter penetration of the peptide into the target cell's membrane (e.g.,through an energy-dependent transport mechanism such as pinocytosis),and/or may lead to an improvement in the peptide's biological activityrelative to the corresponding uncrosslinked (e.g., “unstitched” or“unstapled”) peptide.

Peptides herein may include at least two internally cross-linked orstapled amino acids, wherein the at least two amino acids are separatedby three (i.e., i, i+4) or six (i.e., i, i+7) amino acids. While atleast two amino acids are required to support an internal cross-link(e.g., a staple), additional pairs of internally cross-linked aminoacids can be included in a peptide, e.g., to support additional internalcross-links (e.g., staples). For example peptides can include 1, 2 or 3staples.

Alternatively or in addition, peptides can include three internallycross-linked or stitched amino acids, e.g., yielding two staples arisingfrom a common origin. A peptide stitch includes at least threeinternally cross-linked amino acids, wherein the middle of the threeamino acids forms an internal cross-link (between alpha carbons) witheach of the two flanking (not immediately adjacent) modified aminoacids. The alpha carbon of the core amino acid has side chains that areinternal cross-links to the alpha carbons of other amino acids in thepeptide, which can be saturated or not saturated. Amino acidscross-linked to the core amino acid can be separated from the core aminoacid in either direction by 3 or 6 amino acids. The number of aminoacids on either side of the core (e.g., between the core amino acid andan amino acid cross-linked to the core) can be the same or different.

As noted above an internal tether or cross-link can extend across thelength of one helical turn (i.e., about 3.4 amino acids (i.e., i, i+4)or two helical turns (i.e., about 7 amino acids (i.e., i, i+7).Accordingly, amino acids positioned at i and i+4; or i and i+7 arecandidates for chemical modification and cross-linking. Thus, forexample, where a peptide has the sequence . . . Xaa₁, Xaa₂, Xaa₃, Xaa₄,Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉ . . . , cross-links between Xaa₁ and Xaa₅,or between Xaa₁ and Xaa₈ are useful as are cross-links between Xaa₂ andXaa₆, or between Xaa₂ and Xaa₉, etc.

As disclosed above, peptides herein may include at least one amino acidthat is not one of the 20 common, naturally-occurring amino acids,wherein every position not in that group is a common,naturally-occurring amino acid. In the case of at least one amino acidthat is not one of the 20 common, naturally-occurring amino acids, thepeptide may not have an internal cross-link (or staple). These uncommonamino acids may help maintain the native conformation and/or secondarystructure, particularly under physiologically relevant conditions.

In some instances, peptides can include (e.g., comprise, consistessentially of, or consist of) at least one amino acid, that is not oneof the 20 common, naturally-occurring amino acids, in the at least seven(e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)contiguous amino acids of any of SEQ ID Nos: 1-12.

Peptides can contain one or more asymmetric centers and thus occur asracemates and racemic mixtures, single enantiomers, individualdiastereomers and diastereomeric mixtures and geometric isomers (e.g. Zor cis and E or trans) of any olefins present. For example, peptidesdisclosed herein can exist in particular geometric or stereoisomericforms, including, for example, cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof. Enantiomers can be free(e.g., substantially free) of their corresponding enantiomer, and/or mayalso be optically enriched. “Optically enriched,” as used herein, meansthat the compound is made up of a significantly greater proportion ofone enantiomer. In certain embodiments substantially free means that acomposition contains at least about 90% by weight of a preferredenantiomer. In other embodiments the compound is made up of at leastabout 95%, 98%, or 99% by weight of a preferred enantiomer. Preferredenantiomers may be isolated from racemic mixtures using techniques knownin the art, including, but not limited to, for example, chiral highpressure liquid chromatography (HPLC) and the formation andcrystallization of chiral salts or prepared by asymmetric syntheses(see, e.g., Jacques, et al, Enantiomers, Racemates and Resolutions(Wiley Interscience, New York, 1981); Wilen, S. H., et al., Tetrahedron33:2725 (1977); Eliel, EX. Stereochemistry of Carbon Compounds(McGraw-Hill, N Y, 1962); Wilen, S. H. Tables of Resolving Agents andOptical Resolutions p. 268 (EX. Eliel, Ed., Univ. of Notre Dame Press,Notre Dame, Ind. 1972). All such isomeric forms of these compounds areexpressly included in the present invention.

Peptides can also be represented in multiple tautomeric forms, in suchinstances, the invention expressly includes all tautomeric forms of thecompounds described herein (e.g., isomers in equilibrium (e.g.,keto-enol), wherein alkylation at multiple sites can yieldregioisomers), regioisomers, and oxidation products of the compoundsdisclosed herein (the invention expressly includes all such reactionproducts). All such isomeric forms of such compounds are included as areall crystal forms.

In some instances, the hydrocarbon tethers (i.e., cross links) describedherein can be further manipulated. In one instance, a double bond of ahydrocarbon alkenyl tether, (e.g., as synthesized using aruthenium-catalyzed ring closing metathesis (RCM)) can be oxidized(e.g., via epoxidation or dihydroxylation) to provide one of compoundsbelow.

Either the epoxide moiety or one of the free hydroxyl moieties can befurther functionalized. For example, the epoxide can be treated with anucleophile, which provides additional functionality that can be used,for example, to attach a tag (e.g., a radioisotope or fluorescent tag).The tag can be used to help direct the compound to a desired location inthe body or track the location of the compound in the body.Alternatively, an additional therapeutic agent can be chemicallyattached to the functionalized tether (e.g., an anti-cancer agent suchas rapamycin, vinblastine, taxol, etc.). Such derivatization canalternatively be achieved by synthetic manipulation of the amino orcarboxy-terminus of the polypeptide or via the amino acid side chain.Other agents can be attached to the functionalized tether, e.g., anagent that facilitates entry of the polypeptide into cells.

While hydrocarbon tethers have been described, other tethers are alsoenvisioned. For example, the tether can include one or more of an ether,thioether, ester, amine, or amide moiety. In some cases, a common,naturally-occurring amino acid side chain can be incorporated into thetether. For example, a tether can be coupled with a functional groupsuch as the hydroxyl in serine, the thiol in cysteine, the primary aminein lysine, the acid in aspartate or glutamate, or the amide inasparagine or glutamine. Accordingly, it is possible to create a tetherusing common, naturally-occurring amino acids rather than using a tetherthat is made by coupling two uncommon amino acids. It is also possibleto use a single uncommon amino acid together with a common,naturally-occurring amino acid.

It is further envisioned that the length of the tether can be varied.For instance, a shorter length of tether can be used where it isdesirable to provide a relatively high degree of constraint on thesecondary alpha-helical structure, whereas, in some instances, it isdesirable to provide less constraint on the secondary alpha-helicalstructure, and thus a longer tether may be desired.

Additionally, while examples of tethers spanning from amino acids i toi+3, i to i+4; and i to i+7 have been described in order to provide atether that is primarily on a single face of the alpha helix, thetethers can be synthesized to span any combinations of numbers of aminoacids.

In some instances, alpha disubstituted amino acids are used in thepolypeptide to improve the stability of the alpha helical secondarystructure. However, alpha disubstituted amino acids are not required,and instances using mono-alpha substituents (e.g., in the tethered aminoacids) are also envisioned.

The stapled polypeptides can include a drug, a toxin, a derivative ofpolyethylene glycol; a second polypeptide; a carbohydrate, etc. Where apolymer or other agent is linked to the stapled polypeptide is can bedesirable for the composition to be substantially homogeneous.

The addition of polyethelene glycol (PEG) molecules can improve thepharmacokinetic and pharmacodynamic properties of the polypeptide. Forexample, PEGylation can reduce renal clearance and can result in a morestable plasma concentration. PEG is a water soluble polymer and can berepresented as linked to the polypeptide as formula:

XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y where n is 2 to 10,000 and X is H or aterminal modification, e.g., a C₁₋₄ alkyl; and Y is an amide, carbamateor urea linkage to an amine group (including but not limited to, theepsilon amine of lysine or the N-terminus) of the polypeptide. Y mayalso be a maleimide linkage to a thiol group (including but not limitedto, the thiol group of cysteine). Other methods for linking PEG to apolypeptide, directly or indirectly, are known to those of ordinaryskill in the art. The PEG can be linear or branched. Various forms ofPEG including various functionalized derivatives are commerciallyavailable. PEG having degradable linkages in the backbone can be used.For example, PEG can be prepared with ester linkages that are subject tohydrolysis. Conjugates having degradable PEG linkages are described inWO 99/34833; WO 99/14259, and U.S. Pat. No. 6,348,558.

In certain embodiments, macromolecular polymer (e.g., PEG) is attachedto an agent described herein through an intermediate linker. In certainembodiments, the linker is made up of from 1 to 20 amino acids linked bypeptide bonds, wherein the amino acids are selected from the 20 common,naturally-occurring amino acids. Some of these amino acids may beglycosylated, as is well understood by those in the art. In otherembodiments, the 1 to 20 amino acids are selected from glycine, alanine,proline, asparagine, glutamine, and lysine. In other embodiments, alinker is made up of a majority of amino acids that are stericallyunhindered, such as glycine and alanine. Non-peptide linkers are alsopossible. For example, alkyl linkers such as —NH(CH₂)_(n)C(O)—, whereinn=2-20 can be used. These alkyl linkers may further be substituted byany non-sterically hindering group such as lower alkyl (e.g., C₁-C₆)lower acyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc. U.S. Pat. No.5,446,090 describes a bifunctional PEG linker and its use in formingconjugates having a peptide at each of the PEG linker termini.

The stapled and non-stapled peptides can also be modified, e.g., tofurther facilitate cellular uptake or increase in vivo stability, insome embodiments. For example, acylating or PEGylating a peptidomimeticmacrocycle facilitates cellular uptake, increases bioavailability,increases blood circulation, alters pharmacokinetics, decreasesimmunogenicity and/or decreases the needed frequency of administration.

In some embodiments, the peptides disclosed herein have an enhancedability to penetrate cell membranes.

Methods of synthesizing the compounds of the described herein are knownin the art. Nevertheless, the following exemplary method may be used. Itwill be appreciated that the various steps may be performed in analternate sequence or order to give the desired compounds. Syntheticchemistry transformations and protecting group methodologies (protectionand deprotection) useful in synthesizing the compounds described hereinare known in the art and include, for example, those such as describedin R. Larock, Comprehensive Organic Transformations, VCH Publishers(1989); T. W. Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, 3d. Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser,Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons(1994); and L. Paquette, ed., Encyclopedia of Reagents for OrganicSynthesis, John Wiley and Sons (1995), and subsequent editions thereof.

The peptides of this invention can be made by chemical synthesismethods, which are well known to the ordinarily skilled artisan. See,for example, Fields et al., Chapter 3 in Synthetic Peptides: A User'sGuide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77.Hence, peptides can be synthesized using the automated Merrifieldtechniques of solid phase synthesis with the α-NH₂ protected by eithert-Boc or Fmoc chemistry using side chain protected amino acids on, forexample, an Applied Biosystems Peptide Synthesizer Model 430A or 431.

One manner of making of the peptides described herein is using solidphase peptide synthesis (SPPS). The C-terminal amino acid is attached toa cross-linked polystyrene resin via an acid labile bond with a linkermolecule. This resin is insoluble in the solvents used for synthesis,making it relatively simple and fast to wash away excess reagents andby-products. The N-terminus is protected with the Fmoc group, which isstable in acid, but removable by base. Any side chain functional groupsare protected with base stable, acid labile groups.

Longer peptides could be made by conjoining individual syntheticpeptides using native chemical ligation. Alternatively, the longersynthetic peptides can be synthesized by well-known recombinant DNAtechniques. Such techniques are provided in well-known standard manualswith detailed protocols. To construct a gene encoding a peptide of thisinvention, the amino acid sequence is reverse translated to obtain anucleic acid sequence encoding the amino acid sequence, preferably withcodons that are optimum for the organism in which the gene is to beexpressed. Next, a synthetic gene is made, typically by synthesizingoligonucleotides which encode the peptide and any regulatory elements,if necessary. The synthetic gene is inserted in a suitable cloningvector and transfected into a host cell. The peptide is then expressedunder suitable conditions appropriate for the selected expression systemand host. The peptide is purified and characterized by standard methods.

The peptides can be made in a high-throughput, combinatorial fashion,e.g., using a high-throughput multiple channel combinatorial synthesizeravailable from Advanced Chemtech.

Peptide bonds can be replaced, e.g., to increase physiological stabilityof the peptide, by: a retro-inverso bonds (C(O)—NH); a reduced amidebond (NH—CH₂); a thiomethylene bond (S—CH₂ or CH₂—S); an oxomethylenebond (O—CH₂ or CH₂—O); an ethylene bond (CH₂—CH₂); a thioamide bond(C(S)—NH); a trans-olefin bond (CH═CH); a fluoro substitutedtrans-olefin bond (CF═CH); a ketomethylene bond (C(O)—CHR) or CHR—C(O)wherein R is H or CH₃; and a fluoro-ketomethylene bond (C(O)—CFR orCFR—C(O) wherein R is H or F or CH₃.

The polypeptides can be further modified by: acetylation, amidation,biotinylation, cinnamoylation, farnesylation, fluoresceination,formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyror Thr), stearoylation, succinylation and sulfurylation. As indicatedabove, peptides can be conjugated to, for example, polyethylene glycol(PEG); alkyl groups (e.g., C1-C20 straight or branched alkyl groups);fatty acid radicals; and combinations thereof.

α,α-Disubstituted amino acids containing olefinic side chains of varyinglength can be synthesized by known methods (Williams et al. J. Am. Chem.Soc., 113:9276, 1991; Schafmeister et al., J. Am. Chem Soc., 122:5891,2000; and Bird et al., Methods Enzymol., 446:369, 2008; Bird et al,Current Protocols in Chemical Biology, 2011). For peptides where an ilinked to i+7 staple is used (two turns of the helix stabilized) eitherone S5 amino acid and one R8 is used or one S8 amino acid and one R5amino acid is used. R8 is synthesized using the same route, except thatthe starting chiral auxiliary confers the R-alkyl-stereoisomer. Also,8-iodooctene is used in place of 5-iodopentene. Inhibitors aresynthesized on a solid support using solid-phase peptide synthesis(SPPS) on MBHA resin (see, e.g., WO 2010/148335).

Fmoc-protected α-amino acids (other than the olefinic amino acidsFmoc-S₅—OH, Fmoc-R₈—OH, Fmoc-R₈—OH, Fmoc-S₈—OH and Fmoc-R₅—OH),2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU), and Rink Amide MBHA are commerciallyavailable from, e.g., Novabiochem (San Diego, Calif.). Dimethylformamide(DMF), N-methyl-2-pyrrolidinone (NMP), N,N-diisopropylethylamine (DIEA),trifluoroacetic acid (TFA), 1,2-dichloroethane (DCE), fluoresceinisothiocyanate (FITC), and piperidine are commercially available from,e.g., Sigma-Aldrich. Olefinic amino acid synthesis is reported in theart (Williams et al., Org. Synth., 80:31, 2003).

In some instances, peptides can include a detectable label. As usedherein, a “label” refers to a moiety that has at least one element,isotope, or functional group incorporated into the moiety which enablesdetection of the peptide to which the label is attached. Labels can bedirectly attached (i.e., via a bond) or can be attached by a linker(e.g., such as, for example, a cyclic or acyclic, branched orunbranched, substituted or unsubstituted alkylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted alkenylene; cyclicor acyclic, branched or unbranched, substituted or unsubstitutedalkynylene; cyclic or acyclic, branched or unbranched, substituted orunsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched,substituted or unsubstituted heteroalkenylene; cyclic or acyclic,branched or unbranched, substituted or unsubstituted heteroalkynylene;substituted or unsubstituted arylene; substituted or unsubstitutedheteroarylene; or substituted or unsubstituted acylene, or anycombination thereof, which can make up a linker). Labels can be attachedto a peptide at any position that does not interfere with the biologicalactivity or characteristic of the inventive polypeptide that is beingdetected.

Labels can include: labels that contain isotopic moieties, which may beradioactive or heavy isotopes, including, but not limited to, ²H, ³H,¹³C, ¹⁴C, ¹⁵N, ³¹P, ³²P, ³⁵S, ⁶⁷Ga, ^(99m)TC (Tc-99m), ¹¹¹In, ¹²³I,¹²⁵I, ¹⁶⁹Yb, and ¹⁸⁶Re; labels that include immune or immunoreactivemoieties, which may be antibodies or antigens, which may be bound toenzymes {e.g., such as horseradish peroxidase); labels that are colored,luminescent, phosphorescent, or include fluorescent moieties (e.g., suchas the fluorescent label FITC); labels that have one or morephotoaffinity moieties; labels that have ligand moieties with one ormore known binding partners (such as biotin-streptavidin, FK506-FKBP,etc.).

In some instances, labels can include one or more photoaffinity moietiesfor the direct elucidation of intermolecular interactions in biologicalsystems. A variety of known photophores can be employed, most relying onphotoconversion of diazo compounds, azides, or diazirines to nitrenes orcarbenes (see, e.g., Bayley, H., Photogenerated Reagents in Biochemistryand Molecular Biology (1983), Elsevier, Amsterdam, the entire contentsof which are incorporated herein by reference). In certain embodimentsof the invention, the photoaffinity labels employed are o-, m- andp-azidobenzoyls, substituted with one or more halogen moieties,including, but not limited to 4-azido-2,3,5,6-tetrafluorobenzoic acid.

Labels can also be or can serve as imaging agents. Exemplary imagingagents include, but are not limited to, those used in positron emissionstomography (PET), computer assisted tomography (CAT), single photonemission computerized tomography, x-ray, fluoroscopy, and magneticresonance imaging (MRI); anti-emetics; and contrast agents. Exemplarydiagnostic agents include but are not limited to, fluorescent moieties,luminescent moieties, magnetic moieties; gadolinium chelates (e.g.,gadolinium chelates with DTPA, DTPA-BMA, DOTA and HP-DO3A), ironchelates, magnesium chelates, manganese chelates, copper chelates,chromium chelates, iodine-based materials useful for CAT and x-rayimaging, and radionuclides. Suitable radionuclides include, but are notlimited to, ¹²³I, ¹²⁵I, ¹³⁰I, ¹³¹I, ¹³³I, ¹³⁵I, ⁴⁷Sc, ⁷²As, ⁷²Se, ⁹⁰Y,⁸⁸Y, ⁹⁷Ru, ¹⁰⁰Pd, ¹⁰¹mRh, ¹¹⁹Sb, ¹²⁸Ba, ¹⁹⁷Hg, ²¹¹At, ²¹²Bi, ²¹²Pb,¹⁰⁹Pd, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ⁶⁷Cu, ⁷⁵Br ⁷⁷Br, ⁹⁹mTc, ¹⁴C, ¹³N, ¹⁵O, ³²P,³³P, and ¹⁸F.

Fluorescent and luminescent moieties include, but are not limited to, avariety of different organic or inorganic small molecules commonlyreferred to as “dyes,” “labels,” or “indicators.” Examples include, butare not limited to, fluorescein, rhodamine, acridine dyes, Alexa dyes,cyanine dyes, etc. Fluorescent and luminescent moieties may include avariety of naturally-occurring proteins and derivatives thereof, e.g.,genetically engineered variants. For example, fluorescent proteinsinclude green fluorescent protein (GFP), enhanced GFP, red, blue,yellow, cyan, and sapphire fluorescent proteins, reef coral fluorescentprotein, etc. Luminescent proteins include luciferase, aequorin andderivatives thereof. Numerous fluorescent and luminescent dyes andproteins are known in the art (see, e.g., U.S. Patent Publication2004/0067503; Valeur, B., “Molecular Fluorescence: Principles andApplications,” John Wiley and Sons, 2002; and Handbook of FluorescentProbes and Research Products, Molecular Probes, 9th edition, 2002).

Again, methods suitable for obtaining (e.g., synthesizing), stapling,and purifying the peptides disclosed herein are also known in the art(see, e.g., Bird et. al., Methods in Enzymol., 446:369-386 (2008); Birdet al, Current Protocols in Chemical Biology, 2011; Walensky et al.,Science, 305:1466-1470 (2004); Schafmeister et al., J. Am. Chem. Soc.,122:5891-5892 (2000); U.S. patent application Ser. No. 12/525,123, filedMar. 18, 2010; and U.S. Pat. No. 7,723,468, issued May 25, 2010, each ofwhich are hereby incorporated by reference in their entirety).

In some embodiments, the peptides are substantially free of contaminantsor are isolated. Methods for purifying peptides include, for example,synthesizing the peptide on a solid-phase support. Followingcyclization, the solid-phase support may be isolated and suspended in asolution of a solvent such as DMSO, DMSO/dichloromethane mixture, orDMSO/NMP mixture. The DMSO/dichloromethane or DMSO/NMP mixture maycomprise about 30%, 40%, 50% or 60% DMSO. In a specific embodiment, a50%/50% DMSO/NMP solution is used. The solution may be incubated for aperiod of 1, 6, 12 or 24 hours, following which the resin may be washed,for example with dichloromethane or NMP. In one embodiment, the resin iswashed with NMP. Shaking and bubbling an inert gas into the solution maybe performed.

Pharmaceutical Compositions

One or more of the peptides disclosed herein (e.g., one or more of SEQID NOs: 1-12) can be formulated for use as or in pharmaceuticalcompositions. Such compositions can be formulated or adapted foradministration to a subject via any route, e.g., any route approved bythe Food and Drug Administration (FDA). Exemplary methods are describedin the FDA's CDER Data Standards Manual, version number 004 (which isavailable at fda.give/cder/dsm/DRG/drg00301.htm). For example,compositions can be formulated or adapted for administration byinhalation (e.g., oral and/or nasal inhalation (e.g., via nebulizer orspray)), injection (e.g., intravenously, intra-arterial, subdermally,intraperitoneally, intramuscularly, and/or subcutaneously); and/or fororal administration, transmucosal administration, and/or topicaladministration (including topical (e.g., nasal) sprays and/orsolutions).

In some instances, pharmaceutical compositions can include an effectiveamount of one or more peptides. The terms “effective amount” and“effective to treat,” as used herein, refer to an amount or aconcentration of one or more compounds or a pharmaceutical compositiondescribed herein utilized for a period of time (including acute orchronic administration and periodic or continuous administration) thatis effective within the context of its administration for causing anintended effect or physiological outcome (e.g., treatment of cancer).

Pharmaceutical compositions of this invention can include one or morepeptides and any pharmaceutically acceptable carrier and/or vehicle. Insome instances, pharmaceuticals can further include one or moreadditional therapeutic agents in amounts effective for achieving amodulation of disease or disease symptoms.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, may also beadvantageously used to enhance delivery of compounds of the formulaedescribed herein.

The pharmaceutical compositions of this invention may contain anyconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsor vehicles. In some cases, the pH of the formulation may be adjustedwith pharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated compound or its delivery form. The termparenteral as used herein includes subcutaneous, intra-cutaneous,intra-venous, intra-muscular, intra-articular, intra-arterial,intra-synovial, intra-sternal, intra-thecal, intra-lesional andintra-cranial injection or infusion techniques.

Pharmaceutical compositions can be in the form of a solution or powderfor inhalation and/or nasal administration. Such compositions may beformulated according to techniques known in the art using suitabledispersing or wetting agents (such as, for example, Tween 80) andsuspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose, any blandfixed oil may be employed including synthetic mono- or diglycerides.Fatty acids, such as oleic acid and its glyceride derivatives are usefulin the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents which arecommonly used in the formulation of pharmaceutically acceptable dosageforms such as emulsions and or suspensions. Other commonly usedsurfactants such as Tweens or Spans and/or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutical compositions can be orally administered in any orallyacceptable dosage form including, but not limited to, capsules, tablets,emulsions and aqueous suspensions, dispersions and solutions. In thecase of tablets for oral use, carriers which are commonly used includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions and/or emulsions are administered orally, the activeingredient may be suspended or dissolved in an oily phase is combinedwith emulsifying and/or suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

Alternatively or in addition, pharmaceutical compositions can beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other solubilizing or dispersingagents known in the art.

In some embodiments, the present disclosure provides methods for usingany one or more of the peptides or pharmaceutical compositions(indicated below as ‘X’) disclosed herein in the following methods:

Substance X for use as a medicament in the treatment of one or morediseases or conditions disclosed herein (e.g., cancer, referred to inthe following examples as ‘Y’). Use of substance X for the manufactureof a medicament for the treatment of Y; and substance X for use in thetreatment of Y.

In some instances, one or more peptides disclosed herein can beconjugated, for example, to a carrier protein. Such conjugatedcompositions can be monovalent or multivalent. For example, conjugatedcompositions can include one peptide disclosed herein conjugated to acarrier protein. Alternatively, conjugated compositions can include twoor more peptides disclosed herein conjugated to a carrier.

As used herein, when two entities are “conjugated” to one another theyare linked by a direct or indirect covalent or non-covalent interaction.In certain embodiments, the association is covalent. In otherembodiments, the association is non-covalent. Non-covalent interactionsinclude hydrogen bonding, van der Waals interactions, hydrophobicinteractions, magnetic interactions, electrostatic interactions, etc. Anindirect covalent interaction is when two entities are covalentlyconnected, optionally through a linker group.

Carrier proteins can include any protein that increases or enhancesimmunogenicity in a subject. Exemplary carrier proteins are described inthe art (see, e.g., Fattom et al., Infect. Immun., 58:2309-2312, 1990;Devi et al., Proc. Natl. Acad. Sci. USA 88:7175-7179, 1991; Li et al.,Infect. Immun. 57:3823-3827, 1989; Szu et al., Infect. Immun.59:4555-4561,1991; Szu et al., J. Exp. Med. 166:1510-1524, 1987; and Szuet al., Infect. Immun. 62:4440-4444, 1994). Polymeric carriers can be anatural or a synthetic material containing one or more primary and/orsecondary amino groups, azido groups, or carboxyl groups. Carriers canbe water soluble.

Methods of Treatment

The disclosure includes methods of using the peptides herein for theprophylaxis and/or treatment of cancer. The terms “treat” or “treating,”as used herein, refers to partially or completely alleviating,inhibiting, ameliorating, and/or relieving the disease or condition fromwhich the subject is suffering.

In general, methods include selecting a subject and administering to thesubject an effective amount (a therapeutically effective amount) of oneor more of the peptides herein, e.g., in or as a pharmaceuticalcomposition, and optionally repeating administration as required for theprophylaxis or treatment of a cancer.

Specific dosage and treatment regimens for any particular patient willdepend upon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

EXAMPLES

Described below are studies characterizing variants of MS-1, apreviously described peptide that binds Mcl-1 with increased specificityand affinity relative to Bfl-1, Bcl-xL, Bcl-2, and Bcl-w (Foight et al.ACS Chem. Biol. 2014, 9, 1962-1968). The variants were characterized forsecondary structure, specificity for binding to Mcl-1, affinity ofbinding to Mcl-1, peptide stability, and functionality in cells.Further, a library was created as a tool to discover novel BH3-likepeptides.

Example 1: Characterization of Stapled Variants of MS1

To improve the binding and helicity of MS1 while minimizing itsmolecular weight, we truncated the peptide and generated a stapledvariant by replacing Asn at 4b and Ala at 4f with amino acids containingolefin tethers and performing ruthenium-catalyzed olefin metathesis toyield Mld (see Table 1 for sequences and position notation).Introduction of the hydrocarbon staple at this position of the BH3domain, which we call site “d” in keeping with prior work, has beenshown to promote binding to Mcl-1 (Stewart, M. L.; Fire, E.; Keating, A.E.; Walensky, L. D. Nat Chem Biol 2010, 6 (8), 595). The crystalstructure of a peptide stapled at position d shows direct hydrophobiccontacts made between the staple atoms and the edge of the Mcl-1 bindinggroove.

The secondary structure and binding of Mld were evaluated in solutionusing fluorescence polarization assays and circular dichroism (FIGS. 1aand c ). Mld is −3-fold more helical in solution than MS1. Thehydrocarbon stapling strategy also improved Mcl-1-binding affinity andincreased peptide resistance to proteolysis, in comparison to MS1 (FIGS.1a and d ). Although Mld is able to compete effectively with a peptidecorresponding to the BH3 region of Bim, which is a native partner forMcl-1, structural modeling suggested that Mld may not be maximallyexploiting the available binding opportunities at three helix positions:2e, 3a and 3b. Furthermore, modifying a peptide by introducing a staplechanges its properties, such that re-optimization of the sequence may bebeneficial. However, the virtually unlimited sequence variations thatcould be studied present a challenge. To date, hydrocarbon stapledpeptides have typically been rationally optimized by iterativemutagenesis. Obtaining tight-binding and biologically active moleculesoften requires many rounds of laborious synthesis and stapling ofdifferent peptidic candidates.

The binding curves from representative experiments are shown in FIG. 1.Competition assay of stapled peptides with fluorescently labeled 21merBim-BH3 was performed to measure the binding to Mcl-1 (see FIG. 1 (a)).The standard errors reported are over three experiments. FIG. 1 (b)shows the kinetic analysis of Mld, M2d and M3d binding to Mcl-1, usingbiolayer interferometry. FIG. 1 (c) shows the circular dichroismanalysis of unmodified and stapled variants, in 20 mM tris buffer, pH7.4. HPLC was used to measure the half-lives of unstapled and stapledpeptides exposed to chymotrypsin and the half-lives are shown in FIG. 1(d). BH3 profiling of stapled peptides Mld, M2d, and M3d in Mcl-1 2640cells are shown in FIG. 1 (e). Structural overlay of structures of Mcl-1bound to SAHBd (yellow, PDB 3MK8) and M3d (magenta, model based on 3MK8)can be seen in FIG. 1 (f) and the overlaid structures of MS1 (yellow,model based on 3MK8) and M3d (magenta, also a model based on 3MK8)comparing position 2e is seen in FIG. 1 (g).

TABLE 1 Mcl-1 binding peptide 1F 1G 2A 2B 2C 2D 2E 2F 2G 3A 3B 3C 3D 3E3F 3G 4A 4B 4C 4D 4E 4F 4G 5A MS1 R P E I W M T Q G L R R L G D E I N AY Y A R M1d — — — I W B X Q G L X R L G D E I N A Y Y A R R SEQ ID NO: 2M2d — — — I W B Aib Q G L R R L G D E I X A Y Y X R R SEQ ID NO: 3 M3d —— — I W B Aib Q G Cha R R L G D E I X A Y Y X R R SEQ ID NO: 4 M4d — — —I W B Aib Q G L Q R L G D E I X A Y Y X R R SEQ ID NO: 5 M5d — — — I W BAib Q G L D R L G D E I X A Y Y X R R SEQ ID NO: 6 M6d — — — I W B T Q GL Q R L G D E I X A Y Y X R R SEQ ID NO: 7 M1 — — — I W B T Q G L R R LG D E I N A Y Y A R R Ma-Leu3aCha — — — I W B T Q G Cha R R L G D E I NA Y Y A R R SEQ ID NO: 8 Ma-Leu3aHCha — — — I W B T Q G H-Cha R R L G DE I N A Y Y A R R SEQ ID NO: 9 M1-Thr2eAib — — — I W B Aib Q G L R R L GD E I N A Y Y A R R SEQ ID NO: 10 M1-Leu3aCha/Thr2eAib — — — I W B Aib QG Cha R R L G D E I N A Y Y A R R SEQ ID NO: 11 M1-Leu3aHCha/Thr2eAib —— — I W B Aib Q G H-Cha R R L G D E I N A Y Y A R R SEQ ID NO: 12Table 1. X = an amino acid whose side chain is replaced with anintermolecular link to another amino acid (e.g., α-4-pentenyl alanine),B = Norleucine, Aib = Aminoisobutyric acid, Cha = cyclohexyl alanine,and H-Cha = homo cyclohexyl alanine.

Example 2: Identification of Novel Variants Using One Bead One CompoundLibraries

To accelerate the discovery of therapeutic peptides, we extended theapplication of one-bead-one-compound libraries (Xiao, W.; Bononi, F. C.;Townsend, J.; Li, Y.; Liu, R.; Lam, K. S. Comb. Chem. High ThroughputScreen. 2013, 16 (6), 441) by applying on-bead ring-closing metathesis(RCM) to make libraries of BH3-like stapled peptides (FIG. 5).Installation of a staple into a peptide requires incorporation of twoappropriately spaced α-methyl-α-alkenyl residues, with definedstereochemical configuration and alkene chain length, followed by RCM onthe resin (Kim, Y. W.; Grossman, T. N.; Verdine, G. L. Nat. Protoc.2011, 6 (6), 761). To evaluate conditions that would produce ahigh-quality stapled peptide library suitable for analysis withoutpurification, and to establish an efficient on-bead screening procedure,we synthesized a first-generation stapled library based on the sequenceof Bim BH3 (library LA). This library incorporated a number of mutationsthat have been studied previously to serve as controls. The library wassynthesized on Tentagel macrobeads and screened for binding to Mcl-1 andBcl-xL. Bcl-x_(L) is a paralog of Mcl-1 and an undesired interactiontarget for candidate inhibitors. The screen entailed bead blocking,incubation with biotinylated Mcl-1 (^(Bio)Mcl-1), washing, incubationwith streptavidin-coated quantum dots (Qdots-SA605), and visualizationusing a fluorescence microscope. 173 hit peptides, which were selectedmanually under the microscope, were cleaved from the beads andidentified using mass spectrometry. Both the stapling chemistry andsubsequent tests for binding were carried out with peptides bound toresin, with one unique peptide per bead. Selected hit peptides wereconfirmed to bind to Mcl-1 in solution.

Example 3: Optimization of One Bead One Compound Library

To apply this method to the optimization of Mld, a library of 108stapled peptides (library LB) was designed to be enriched in Mcl-1binders. Positions 2e, 3a and 3b were diversified with 3, 4 and 9 aminoac-ids, respectively, varying the residue size, hydrophobicity and/orcharge (Table 1). Many of the side chains were chosen to be hydrophobicto favor hydrophobic contacts with Mcl-1. We also introduced knownhelix-inducing side chains to improve the helical content of stapledpeptides, which is expected to be poor at the peptide N-terminus becauseof two Gly residues in the MS1 template. To choose residues for themutation sites, we used the large amount of mutational data from SPOTarrays and side-chain scanning of BH3 peptides (Dutta, S.; Guild, S.;Chen, T. S.; Fire, E.; Grant, R. A.; Keating, A. E. J. Mol. Biol. 2010,398 (5), 747; Foight, G. W.; Ryan, J. A.; Gullá, S. V; Letai, A.;Keating, A. E. ACS Chem. Biol. 2014, 9 (9), 1962; Boersma, M. D.;Sadowsky, J. D.; Tomita, Y. A.; Gellman, S. H. Protein Sci. 2008, 17(7), 1232). We also analyzed structures of Mcl-1 bound to different BH3variants using Bioluminate (version 1.9, Schrödinger, LLC, New York,N.Y., 2015) to select side chains for the library. We incorporatedfunctionalities, beyond those found in the 20 common,naturally-occurring amino acids, that we predicted could access crevicesin Mcl-1 that are too distant to be reached by the sidechains found inthe 20 common, naturally-occurring amino acids. LB was sorted forbinding to Mcl-1 in two rounds of competition screening in which beadswere selected that could bind to Mcl-1 in the presence of 12.5- or50-fold higher con-centration of Bcl-xL.

Example 4: Screening of Optimized Library

LB was initially screened under conditions optimized for the firstgeneration library, LA: 0.2 μM ^(Bio) Mcl-1 in presence of 2.5 μMmyc-tagged Bcl-xL (^(myc)Bcl-xL). However, for LB we used a ComplexObject Parametric Analyzer and Sorter (COPAS) for cell sorting thatpermits fluorescence-based sorting of up to 300 beads/s. The 5% of beadswith highest fluorescence intensities were sorted into 96-well plates.Beads were washed and then post-screened using more stringentconditions: 0.05 μm ^(Bio)mcl-1 plus 2.5 μM ^(myc)Bcl-xl. Afterincubation with streptavidin-coated quantum dots, beads were furtherwashed and visualized using a fluorescence microscope. 170 brightestbeads from a pool of −10,000 were isolated manually. MALDI massspectrometry confirmed that the selected beads included at least 6different sequences out of the possible 108 stapled BH3 peptides.Sequences M1d (32 beads), M2d (48 beads), and M3d (38 beads) were chosenfor further analysis (Table 1). Interestingly, M2d and M3d incorporatepreviously untested side chains at positions 2e and 3a, which are highlyconserved in known BH3 motifs.

Example 5: Characterization of M2d and M3d Peptide Binding to Mcl-1

M2d and M3d were tested in solution in competition with a fluorescentlylabeled Bim BH3 peptide for binding to five human Bcl-2 paralogs (FIG. 1and FIG. 2). Unlabeled peptides were mixed with fluoresceinated Bim BH3and one of Mcl-1, Bfl-1, Bcl-w, Bcl-xL, or Bcl-2. The competitionexperiments indicated that M2d and M3d are both highly selective forMcl-1 over 4 other anti-apoptotic members and are both considerablytighter binders of Mcl-1 than is M1d. These peptides competed withfluoresceinated Bim 21mer for Mcl-1 binding with half-maximal inhibitoryconcentrations (IC₅₀ values) of 106±12 nM and 72±11 nM, for M2d and M3drespectively (compare with 350 nM for M1d and 811 nM for MS1). Position2e is usually conserved as small (alanine, glycine, serine) in naturalBH3 sequences. However, previous studies have shown that Mcl-1 can bindBH3 peptides with bulkier threonine and leucine at this site (Foight, G,W.; Ryan, J. A.; Gulla, S. V; Letai, A.; Keating, A. E. ACS Chem. Biol.2014, 9(9), 1962 and Stewart, M. L.; Fire, E.; Keating, A. E.; Walensky,L. D. Nat Chem Biol 2010, 6 (8), 595). Our results show that, likewise,introducing the larger, branched 2-aminoisobutyric acid (Aib) at the 2eposition in M2d is not only well tolerated but increases bindingaffinity for Mcl-1. Computational modeling of Aib at 2e in Mcl-1complexes (2PQK and 3MK8) shows how two methyl groups can beaccommodated in the Mcl-1 interface (FIG. 1(g)). Interestingly, thelargest increase in potency was observed for M3d, which includes Aib at2e and Cha at 3a. The selection of Cha at 3a was not easily anticipatedfrom the inspection of Mcl-1 complexed with BH3 peptides, and thestabilizing effect was somewhat surprising given that leucine is highlyconserved at this position in native BH3 domains. Molecular modelingusing Bioluminate predicts that the flexible cyclohexyl moiety of M3dpenetrates deep into the previously defined P2 binding pocket of Mcl-1(FIG. 1(f)), explaining the increase in potency of M3d for Mcl-1 overother Bcl-2 paralogs (FIG. 1(a))(Burke, J. P.; Bian, Z.; Shaw, S.; Zhao,B.; Goodwin, C. M.; Belmar, J.; Browning, C. F.; Vigil, D.; Friberg, A.;Camper, D. V; Rossanese, 0. W.; Lee, T.; Olejniczak, E. T.; Fesik, S. W.J. Med. Chem. 2015, 58 (9), 3794). Discovery of unpredictable chemicalmoieties strongly improving Mcl-1 binding clearly demonstrate theeffectiveness of our combinatorial approach.

We used biolayer interferometry to measure the kinetics of binding ofstapled peptides to Mcl-1 (FIG. 1(b)). We generated biotinylated stapledpeptides and attached these to a streptavidin-modified probe surface.Binding of M2d and M3d to Mcl-1 was slower compared to binding of Mld,likely due to rearrangements required for packing of the bulky sidechains of Aib and Cha. However, consistent with their higher affinity,dissociation of M2d and M3d from Mcl-1 was even slower compared to Mld.Peptide M3d had the lowest k_(diss) (1.8×10⁻⁴) and K_(d) (5 nM) values.

Example 6: Structural Characterization of Mld, M2d and M3d Peptides

The secondary structure of selected stapled peptides was assessed bycircular dichroism (CD) spectroscopy (FIG. 1(c)). Interestingly, theobserved trend for binding affinities was paralleled by the measuredhelicity. M2d and M3d showed significantly higher helicity than Mld,with mean residue ellipticity (MRE) at 222 nm of 15,450 and 21,000 degcm² dmol⁻1, respectively. Therefore, the increases in helicity can beattributed to both helix-inducing substitutions: Aib at 2e and Cha at 3a(Armstrong, K. M.; Fairman, R.; Baldwin, R. L. J. Mol Biol. 1993, 230(1), 284. and Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Chem.Rev. 2001, 101 (10), 3131).

Example 7: Characterization of Mld, M2d and M3d Peptide Stability

All of the stapled MS1 variants that we tested were more proteaseresistant than un-metathesized Mld, which is a key pharmacologicadvantage of the stapling approach. Unstapled Mld has a half-life2-6-fold shorter than the corresponding stapled peptides (FIG. 1(d)).Notably, the protease resistance analysis revealed longer half-lives forboth M2d and M3d compared to Mld, indicating the utility of introducinghelix-promoting amino acids into a stapled peptide for maximizingprotease resistance.

Example 8: BH3 Profiling of Peptide Variants

We performed BH3 profiling to test the function of stapled variants ofMS1 in cells. A whole-cell BH3 profiling experiment quantifies thedependence of cancer cell mitochondrial integrity on specificanti-apoptotic proteins and can be predictive of cellular responses tochemotherapy (Ryan, J. A.; Brunelle, J. K.; Letai, A. Proc. Natl. Acad.Sci. 2010, 107 (29), 12895 and Ryan, J. A; Letai, A. Methods 2013,61(2), 156). In this assay, permeabilized cells are stained with dye JC1to monitor mitochondrial membrane integrity in response to increasingdoses of BH3 peptides. We used this assay to test the specificity of ourMcl-1-binding stapled peptides in cell lines with different establisheddependencies on Bcl-x_(L) and Mcl-1. Mcl-1/Myc 2640 is an engineeredmurine leukemia cell line overexpressing murine Mcl-1 and Myc, andMDA-MB 231 is a human breast cancer cell line with a primedBcl-xL-dependent pro-file (Brunelle, J. K.; Ryan, J.; Yecies, D.;Opferman, J. T.; Letai, A. J. Cell Biol. 2009, 187 (3), 429 and Ryan, J.A.; Brunelle, J. K.; Letai, A. Proc. Natl. Acad. Sci. 2010, 107 (29),12895). By BH3 profiling using native BH3 peptides from BAD and NOXAA,we confirmed cell line dependencies on these anti-apoptotic proteins(FIG. 3). FIG. 3 shows the BH3 profiling of cell lines using engineered(MS1) and native BH3 peptides (Bim, NoxA). Mcl-1 2640 cell line isdependent on Mcl-1 whereas MDA-MB-231 is dependent on Bcl-xL, asindicated by response to NoxA and Bad, respectively. BBDL is Bax/Bakdeficient leukocyte (error bars indicate the standard deviation over 3or more replicates). Both Mld and M2d showed Bax/Bak independentactivity in Bax/Bak deficient cells, indicating non-specific toxicity(FIG. 4). However, peptide M3d showed no activity in Bax/Bak negativecells, consistent with an on-target mechanism, (Deng, J.; Carlson, N.;Takeyama, K.; Dal Cin, P.; Shipp, M.; Letai, A. Cancer Cell 2015, 12(2), 171) and was remarkably potent when tested on Mcl-1/Myc 2640, withan EC₅₀ value of 30 nM (FIGS. 1(e) and 4(b). The specificity of M3d wasconfirmed by the much higher EC₅₀ (estimated as >5 mM) observed for theBcl-x_(L) dependent cell line MDA-MB 231 (FIG. 4(a)).

Example 9: Characterization of Novel Non-Stapled Peptides with AminoAcids that are not the 20 Common, Naturally-Occurring Amino Acids

We identified two substitutions that—both in the presence or absence ofa staple modification—improve binding to Mcl-1 and preserve a highdegree of specificity. These modifications are: replacement of a leucineresidue at position “3a” with either cyclohexyl phenylalanine orhomo-cyclohexyl phenylalanine and replacement of a threonine at position“2e” with 2-amino isobutyric acid (see Table 1). Both arenon-conservative mutations, and the leucine that is substituted in ourpeptides is highly conserved in natural peptides that bind to Mcl-1,making it somewhat surprising that a change to the larger cyclohexylphenylalanine or homo-cyclohexyl phenylalanine groups is beneficial.FIG. 6 shows the sequences of both stapled and non-stapled peptides,along with the structures of various uncommon amino acid examples. FIGS.7 and 8, along with the following describe the binding and biophysicalcharacterization of the new staple-less peptides.

The secondary structure of selected non-stapled peptides with uncommonamino acids was assessed by circular dichroism (CD) spectroscopy at 22°C. in H₂O (FIG. 7). Single point mutations were compared with theirparent construct (M1). Increase in helical content can be attributed tothe addition of helix-inducing amino acids.

The binding affinity and specificity for the non-stapled peptides withuncommon amino acids were assessed using a competition assay. IC₅₀values for binding to Mcl-1 (target) or Bcl-xL (undesired competitor)were obtained from competition assays with fluoresceinated Bim peptide(FIG. 8). All single point mutations incorporated conferred higherbinding affinity to Mcl-1 and preserve selectivity over other Bcl-2family proteins. Data are the mean and s.d. for at least duplicateexperiments.

Example 10: BH3 Profiling of Novel Non-Stapled Peptides

We measured the depolarization of the mitochondrial membrane of p185+B-ALL cell lines dependent on Mcl-1, Bcl-xL, Bcl-2 or Bfl-1 in responseto treatment with unstapled peptides in comparison with MS1. The resultsfor the peptides compared to MS1 at 10 nM are shown in the top panel ofFIG. 9. The results of known BH3 peptides at a higher concentration (10μM) are shown in the bottom panel of FIG. 9. In the sequences listed,1=Aib, 2-aminoisobutyric acid; 2=Cha, cyclohexylalanine; and 3=h-Cha,homo-cyclohexylalanine.

The unstapled peptides in the top panel of FIG. 9 were highly potent andselective for inducing MOMP in Mcl-1 dependent, but not Bcl-xL, Bcl-2 orBfl-1 dependent permeabilized cells at 10 nM. The known peptides (BIM,PUMA, BAD, NOXAA) were less potent and were tested at 10 μM, as shown inthe bottom panel of FIG. 9; these peptides were less selective. Theoverexpressing cell lines are described in: Koss et al. “Definingspecificity and on-target activity of BH3-mimetics using engineeredB-ALL cell lines.” Oncotarget (2016) vol. 7 (10) pp. 11500-11, which isincorporated herein in its entirety.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A compound comprising the amino acid sequence: 1F 1G 2A 2B 2C 2D 2E2F 2G 3A 3B 3C 3D 3E 3F 3G 4A 4B 4C 4D 4E 4F 4G 5A (SEQ ID NO: 1),wherein 1F is R or a conservative substitution or is missing; 1G is P ora conservative substitution or is missing; 2A is E or a conservativesubstitution or is missing; 2B is I or a conservative substitution; 2Cis W or a conservative substitution; 2D is M or a conservativesubstitution, or norleucine (B); 2E is T or a conservative substitution,V or a conservative substitution, 2-aminoisobutyric acid (Aib); 2F is Qor a conservative substitution; 2G is G or a conservative substitution;3A is L or a conservative substitution, F or a conservativesubstitution, pentafluoro phenylalanine, cyclohexyl alanine (Cha), orhomo-cyclohexyl alanine (H-Cha); 3B is R or a conservative substitution,W or a conservative substitution, Q or a conservative substitution, D ora conservative substitution, Y or a conservative substitution, Aib,D-phenyl glycine, α,α methyl leucine, α,α methyl phenylalanine; 3C is Ror a conservative substitution; 3D is L or a conservative substitution;3E is G or a conservative substitution; 3F is D or a conservativesubstitution; 3G is E or a conservative substitution; 4A is I or aconservative substitution; 4B is N or a conservative substitution; 4C isA or a conservative substitution; 4D is Y or a conservativesubstitution; 4E is Y or a conservative substitution; 4F is A or aconservative substitution; 4G is R or a conservative substitution or ismissing; 5A is R or a conservative or is missing; provided that 2E, 3A,3B, 4B and 4F are not T, L, R, N and A respectively, and whereinoptionally, the side chains of two amino acids separated by 3 or 6 aminoacids are replaced by an intermolecular crosslink. 2.-62. (canceled)